scholarly journals Cytokinins Stimulate Plasmodesmatal Transport in Leaves

2021 ◽  
Vol 12 ◽  
Author(s):  
Wilson Horner ◽  
Jacob O. Brunkard
Keyword(s):  
Cell Walls ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Plant Biology ◽  
Negative Regulator ◽  
Cytokinin Signaling ◽  
Dynamic Changes ◽  
Cytokinin Receptors ◽  
Green Fluorescent ◽  
Development Regulation

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1–4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Janet M Murray ◽  
Douglas I Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fluorescent Protein ◽  
Amino Acid Identity ◽  
Negative Regulator ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Isolation And Characterization ◽  
Nucleotide Exchange Factor ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.

207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 212
Author(s):  
C. A. Pinzon ◽  
M. Snyder ◽  
J. Pryor ◽  
B. Thompson ◽  
M. Golding ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Muscle Mass ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Negative Regulator ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells

2012 ◽  
Vol 77 (6) ◽  
pp. 603-608 ◽  
Author(s):  
T. V. Komarova ◽  
E. V. Sheval ◽  
D. V. Pozdyshev ◽  
V. S. Kolesnikova ◽  
Yu. L. Dorokhov
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Green Fluorescent Protein Production ◽  
Green Fluorescent

scholarly journals FoSTUA, Encoding a Basic Helix-Loop-Helix Protein, Differentially Regulates Development of Three Kinds of Asexual Spores, Macroconidia, Microconidia, and Chlamydospores, in the Fungal Plant Pathogen Fusarium oxysporum

2004 ◽  
Vol 3 (6) ◽  
pp. 1412-1422 ◽  
Author(s):  
Toshiaki Ohara ◽  
Takashi Tsuge
Keyword(s):  
Fusarium Oxysporum ◽  
Fluorescent Protein ◽  
Negative Regulator ◽  
Vascular Wilt ◽  
Wild Type ◽  
Low Frequencies ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Chlamydospore Formation ◽  
Green Fluorescent

ABSTRACT The soil-borne fungus Fusarium oxysporum causes vascular wilt of a wide variety of plant species. F. oxysporum produces three kinds of asexual spores, macroconidia, microconidia, and chlamydospores. Falcate macroconidia are formed generally from terminal phialides on conidiophores and rarely from intercalary phialides on hyphae. Ellipsoidal microconidia are formed from intercalary phialides on hyphae. Globose chlamydospores with thick walls are developed by the modification of hyphal and conidial cells. Here we describe FoSTUA of F. oxysporum, which differentially regulates the development of macroconidia, microconidia, and chlamydospores. FoSTUA encodes a basic helix-loop-helix protein with similarity to Aspergillus nidulans StuA, which has been identified as a transcriptional regulator controlling conidiation. Nuclear localization of FoStuA was verified by using strains expressing FoStuA-green fluorescent protein fusions. The FoSTUA-targeted mutants exhibited normal microconidium formation in cultures. However, the mutants lacked conidiophores and produced macroconidia at low frequencies only from intercalary phialides. Thus, FoSTUA appears to be necessary to induce conidiophore differentiation. In contrast, chlamydospore formation was dramatically promoted in the mutants. These data demonstrate that FoStuA is a positive regulator and a negative regulator for the development of macroconidia and chlamydospores, respectively, and is dispensable for microconidium formation in cultures. The disease-causing ability of F. oxysporum was not affected by mutations in FoSTUA. However, the mutants produced markedly fewer macroconidia and microconidia in infected plants than the wild type. These results suggest that FoSTUA also has an important role for microconidium formation specifically in infected plants.

Role of Golgi Apparatus in the Formation of Plant Slimes, Cuticles and Extraneous Wall Material

1974 ◽  
Vol 32 ◽  
pp. 86-87
Author(s):  
Hilton H. Mollenhauer
Keyword(s):  
Cell Wall ◽  
Golgi Apparatus ◽  
Cell Walls ◽  
Growth And Development ◽  
Characteristic Property ◽  
Plant Cells ◽  
Plant Biology ◽  
Wall Material ◽  
Wall Properties

Cell walls are fundamentally involved in many aspects of plant biology including the morphology, growth, and development of plant cells and the interactions between plant hosts and their pathogens. Intuitively, one can recognize that these wall properties result from the sum total of the various components of which the wall is composed and that there are classes of substances each of which impart a characteristic property to the cell wall.

scholarly journals Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin

2009 ◽  
Vol 418 (3) ◽  
pp. 615-624 ◽  
Author(s):  
Philip Bardelang ◽  
Mireille Vankemmelbeke ◽  
Ying Zhang ◽  
Hannah Jarvis ◽  
Eleni Antoniadou ◽  
...  
Keyword(s):  
Cell Walls ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Target Site ◽  
Protein Domain ◽  
Serine Residue ◽  
Endopeptidase Activity ◽  
Green Fluorescent ◽  
Colicin E9

We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

1999 ◽  
Vol 18 (9) ◽  
pp. 707-714 ◽  
Author(s):  
A. R. Elliott ◽  
J. A. Campbell ◽  
B. Dugdale ◽  
R. I. S. Brettell ◽  
C. P. L. Grof
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
In Vivo Detection ◽  
Green Fluorescent
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